WO2011137445A2 - Analyse par amplification en chaîne par polymérase d'un échantillon de salive pour détecter une infection à cytomégalovirus - Google Patents

Analyse par amplification en chaîne par polymérase d'un échantillon de salive pour détecter une infection à cytomégalovirus Download PDF

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WO2011137445A2
WO2011137445A2 PCT/US2011/034809 US2011034809W WO2011137445A2 WO 2011137445 A2 WO2011137445 A2 WO 2011137445A2 US 2011034809 W US2011034809 W US 2011034809W WO 2011137445 A2 WO2011137445 A2 WO 2011137445A2
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seq
cmv
probe
saliva
kit
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PCT/US2011/034809
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WO2011137445A3 (fr
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Suresh Boppana
Zdenek Novak
Shannon Ross
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The Uab Research Foundation
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Publication of WO2011137445A3 publication Critical patent/WO2011137445A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the field of the disclosure is assays to detect nucleic acids, particularly those to detect nucleic acids indicative of the presence of a pathogen in a biological sample.
  • Cytomegalovirus is one of the herpesviruses. This group of viruses includes the herpes simplex viruses, varicella-zoster virus (which causes chickenpox and shingles), and Epstein-Barr virus (which causes infectious mononucleosis, also known as mono).
  • CMV is a common virus that infects people of all ages. Most people with CMV infections have no signs or symptoms. However, CMV can cause disease in people with a weakened immune system such as transplant patients, people infected with HIV, and newborns; newborn babies are by far the largest group vulnerable to CMV. About 1 in 150 children is born with congenital CMV infection.
  • CMV can be transmitted from a pregnant woman to her fetus during pregnancy.
  • the virus in the mother's blood crosses over the placenta and infects the fetus' blood.
  • Most babies with congenital CMV infection never have health problems.
  • congenital CMV infection causes health problems that may be apparent at birth or may develop later during infancy or childhood.
  • serious symptoms of CMV infection are present at birth, such as prematurity, liver problems, lung problems, spleen problems, low birth weight, small head size (microcephaly), and seizures.
  • Congenital CMV can cause chronic illness, leading to hearing loss, vision loss, mental disability, small head size, lack of physical coordination, seizures, and death.
  • CMV is a leading non-genetic cause of sensorineural hearing loss (SNHL). About 10% to 15% of children born with congenital CMV infection develop SNHL, although most do not exhibit symptoms at birth.
  • PCR polymerase chain reaction
  • a general embodiment of the method comprises contacting the sample with a first forward amplification primer and a first reverse amplification primer, said primers hybridizing under highly stringent conditions to sequences flanking a first target sequence, to form a reaction mixture; performing the polymerase chain reaction on the reaction mixture to form an amplicon; and detecting the target sequence in the amplicon; wherein the sample is not subject to DNA extraction prior to performing the polymerase chain reaction.
  • An alternative general embodiment of the method comprises contacting the sample with a first forward amplification primer and a first reverse amplification primer, said primers hybridizing under highly stringent conditions to sequences flanking a target sequence; contacting the sample with a second forward amplification primer and a second reverse amplification primer, said second primers hybridizing under highly stringent conditions to sequences flanking a second target sequence, to form a reaction mixture; performing the polymerase chain reaction on the reaction mixture to form an amplicon; and detecting the target sequence in the amplicon.
  • the target sequence in the amplicon is detected through the use of a probe that hybridizes specifically to the target sequence under highly stringent conditions. Additional forward and reverse primers may be added as desired to amplify additional targets within the amplicon.
  • a particular embodiment of the method comprises contacting a saliva sample with a first forward amplification primer and a first reverse amplification primer, said primers hybridizing under highly stringent conditions to the major envelope glycoprotein B gene or a portion thereof; contacting the sample with a second forward amplification primer and a second reverse amplification primer, said second primers hybridizing under highly stringent conditions to sequences flanking the immediate early region 2 (IE-2) exon 5 or a portion thereof, to form a reaction mixture; performing real-time polymerase chain reaction on the reaction mixture to form an amplicon; contacting the amplicon with a probe specific for the amplified region of glycoprotein B under highly stringent conditions; contacting the amplicon with a probe specific to the amplified regions of IE-2 exon 5 under highly stringent conditions; and detecting any hybridized probe.
  • IE-2 immediate early region 2
  • kits for the detection of a virus in saliva by real-time PCR are provided.
  • the kit comprises a first forward primer, a first reverse primer, a DNA polymerase, and does not include reagents for DNA extraction.
  • the kit comprises a first forward primer, a first reverse primer, a second forward primer, a second reverse primer, and a DNA polymerase.
  • the kit is for the detection of human CMV in a saliva sample
  • the first primers are specific for the major envelope glycoprotein B or a portion thereof
  • the second primers are specific for the immediate IE- 2 exon 5 or a portion thereof
  • the kit further comprises a first probe specific for the amplified region of the major envelope glycoprotein B
  • the kit further comprises a second probe specific for the IE-2 exon 5.
  • FIG 1. A diagram illustrating prospective screening study of 34,989 newborns to determine the sensitivity and specificity of saliva real-time PCR assays in identifying infants with congenital CMV infection.
  • FIG. 2 A simplified map of the human cytomegalovirus genome. UL - unique long, US - unique short, TRL - terminal repeat long, IRL - internal repeat long (inverted repeat of TRL), TRS -terminal repeat short, IRS - internal repeat short (inverted repeat of TRS). From NIST, citing Kotenko et al. PNAS 97: 1695-1700 (2000).
  • FIG. 3 A detailed map of the human cytomegalovirus genome.
  • the green arrows represent previously annotated ORFs of HCMV deemed likely to encode a bona fide polypeptide, red arrows designate ORFs deemed unlikely to encode a polypeptide, and blue arrows indicated previously unrecognized ORFs that the author's analysis predicts have high potential to encode proteins.
  • the gray box marks the additional sequence found in the HCMV Toledo strain, locating it with respect to the AD 169 genome. Rectangles superimposed on the line represent the sequence-identify terminal repeats. Each mark on the sequence line represents 1 ,000 bp. From NIST, citing Murphy et al. PNAS 100 (2003).
  • FIG 4 Organization of open reading frames in several strains of human CMV.
  • A Conventional ORF maps of the AD 169 laboratory strain and clinical isolates. From the left, the AD169 genome contains TRL1-14 (green arrow), UL1-132 (dark blue arrow), IRL14-1 (green arrow), IRS1 (red arrow), US 1-36 (light blue arrow), and TRS1 (red arrow). From the left, clinical isolates contain a unique domain including RL1-14 and UL1-151 (green plus dark blue arrow) followed by IRS1 (red arrow), US 1-36 (light blue arrow), and TRS 1 (red arrow).
  • B ORF maps of the BAC clones whose sequences are reported in Murphy et al. (see citation below).
  • Sequences were linearlized at the position corresponding to nucleotide 1 of the original AD 169 sequence. Arrows indicate the relative orientations of the repeated and unique ORF blocks.
  • the RL region is not repeated in the clinical isolates (Toledo, PH, TR, and FIX); there is a single copy of the region (green segment appended to the unique long region).
  • the Towne laboratory strain contains a block of ORFs (UL147-154, orange arrow) that is not present in AD 169, and the clinical isolates contain a block of ORFs (UL133-151, orange arrow) that is not present in the AD169 laboratory strains.
  • the BAC inserts are identified (B), and viral ORFs deleted during BAC insertions are listed in parentheses. From NIST, citing Murphy et al. PNAS 100 no 25 (2003).
  • highly stringent conditions means that the conditions of temperature and ionic strength are selected so that it enables hybridization to be maintained between two complementary nucleic acid fragments. Such conditions can also be affected by the presence of certain enzymes, notably helicase. These conditions are well known by the person skilled in the art, and are described, for example, in the book by Sambrook at el. Molecular Cloning, a Laboratory Manual. Third Edition. CSHL press, Cold Spring Harbor, New York, 2001 , which is incorporated herein by reference to teach how to achieve such conditions. A person of ordinary skill in the art would be able to fluctuate the various factors involved to achieve the desired level of stringency for a given pair of complementary DNA fragments with a known melting point.
  • hybridization of two strands at high stringency requires that the sequences exhibit a high degree of complementarity over an extended portion of their length.
  • high stringency conditions include: hybridization to filter-bound DNA in 0.5 M NaHP0 4 , 7% SDS, 1 mM EDTA at 65° C, followed by washing in 0.1 x SSC/0.1% SDS (where 1 x SSC is 0.15 M NaCl, 0.15 M Na citrate) at 68° C or for oligonucleotide molecules washing in 6 x SSC/0.5% sodium pyrophosphate at about 37° C (for 14 nucleotide-long oligonucleotides), at about 48° C (for about 17 nucleotide-long oligonucleotides), at about 55° C (for 20 nucleotide-long oligonucleotides), and at about 60° C (for 23 nucleotide-long oligonucleotides).
  • the highly stringent include: hybridization to filter
  • Conditions of intermediate or moderate stringency such as, for example, an aqueous solution of 2 x SSC at 65° C; alternatively, for example, hybridization to filter-bound DNA in 0.5 M NaHP0 4 , 7% SDS, 1 mM EDTA at 65° C followed by washing in 0.2 x SSC/0.1% SDS at 42° C
  • low stringency such as, for example, an aqueous solution of 2 x SSC at 55° C
  • Specific temperature and salt conditions for any given stringency hybridization reaction depend on the concentration of the target DNA and length and base composition of the probe, and are normally determined empirically in preliminary experiments, which are routine (see Southern, J. Mol. Biol.
  • standard hybridization conditions refers to hybridization conditions that allow hybridization of two nucleotide molecules having at least 50% sequence identity. According to a specific embodiment, hybridization conditions of higher stringency may be used to allow hybridization of only sequences having at least 75% sequence identity, at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity.
  • the term "individual”, “subject” or “patient” as used herein refers to any animal, including birds or mammals, such as mice, Norway rats, cotton rats, gerbils, cavies, hamsters, other rodents, rabbits, dogs, cats, swine, cattle, sheep, goat, horses, domestic fowl, primates, and humans.
  • the term may specify male or female or both, or exclude male or female.
  • wild-type refers to an allele of a gene that is capable of providing the normal functioning of the gene and is the predominant allele in the population.
  • mutant refers to an allele that is not wild-type. Some mutant alleles are phenotypically similar or indistinguishable from the wild- type, while other mutant alleles produce a phenotype that is easily distinguished from that associated with the wild-type.
  • a method for the detection of viruses comprises performing a genetic analysis on a sample. Some embodiments of the method comprise detecting CMV in a sample using PCR. Some such embodiments of the genetic analysis comprise the polymerase chain reaction utilizing primers that will hybridize under highly stringent conditions with at least one of a nucleic acid consensus sequence for CMV, a sequence unique to CMV, or a region flanking such a sequence.
  • the sample may be any material that is suspected to contain the virus. Examples of such samples include a biological sample, a sample from a subject, an environmental sample, and a culture sample.
  • the sample is a biological sample from a subject, including a fluid or tissue sample.
  • the fluid or tissue may be any that is known or suspected to harbor the virus.
  • the subject may be any organism that is known or suspected to harbor the virus, including Homo sapiens.
  • the subject is a human neonate.
  • Some embodiments of the sample comprise a saliva sample, for example obtained from a cheek swab.
  • the sample is dried to stabilize the sample during storage or transportation.
  • the sample is stored in a liquid medium, including a sucrose/phosphate transport medium (TM).
  • TM sucrose/phosphate transport medium
  • the virus may be any virus suspected to exist in the sample.
  • the virus is a virus that is found in saliva.
  • the virus is CMV.
  • the CMV may be a human CMV or a CMV found in another animal.
  • the CMV is a specific strain of human CMV.
  • the sample suspected of harboring the virus can be analyzed.
  • the analysis may be performed using any techniques known in the art including, but not limited to, sequencing, PCR, RT-PCR, quantitative PCR, restriction fragment length polymorphism, hybridization techniques, Northern blot, microarray technology, and similar techniques.
  • the level of expression may be normalized by comparison to the expression of another gene such as a well known, well characterized gene or a housekeeping gene.
  • reverse-transcriptase PCR can be used to detect the presence of a specific mRNA population in a complex mixture of thousands of other mRNA species.
  • Hybridization to clones or oligonucleotides arrayed on a solid support can be used to both detect the presence of and quantitate the level of a target sequence.
  • a target sequence complementary to a sequence encoding a target sequence (which may be referred to as an analyte) to be detected is fixed to a substrate.
  • the substrate may be of any suitable type including but not limited to glass, nitrocellulose, nylon or plastic. At least a portion of the complementary nucleic acid is attached to the substrate and then incubated with the analyte, isolated from the sample of interest.
  • Hybridization between the substrate bound nucleic acid and the analyte can be detected and quantitated by several means including but not limited to radioactive labeling or fluorescence labeling of the analyte or a secondary molecule designed to detect the hybrid. Quantitation of the target sequence can be done by comparison of the intensity of the signal from the analyte compared with that determined from known standards.
  • PCR polymerase chain reaction
  • Variants of PCR may be used, including real-time (quantitative) PCR.
  • Other suitable variant of PCR may be used with the assay, including quantitative PCR, multiplex PCR, nested PCR, multiplex ligation-dependent probe amplification, miniprimer PCR, and hot-start PCR.
  • Loop-mediated isothermal amplification (LAMP) can be used, as described by Notoni et al., Nucleic Acid Res.
  • LAMP has been used successfully to detect CMV in vitreous fluid, for example (Reddy et al, J. Clinical Microbiol, 48(6):2050-2052 (2010)).
  • Real-time PCR has the advantage of allowing direct and immediate observation of target amplification, enhancing the sensitivity of the method and reducing the time required.
  • the specificity of the hybridization between the primer and the target sequence can be adjusted by increasing or decreasing the length of the primer.
  • a primer will give increased specificity and differentiation as a mismatch between the target sequence and the primer will have a significant impact on the hybridization efficiency.
  • a longer primer will provide less specificity but greater hybridization efficiency and therefore increased sensitivity.
  • the nature of the primer will influence the composition of the primer.
  • One of ordinary skill in the art would be able to alter the parameters of the primer to achieve the desired specificity and sensitivity of binding of the primer to the target sequence.
  • the primer is 10 to 50 base pairs in length.
  • the primer is 20-40 base pairs in length.
  • the primer is 15 to 26 base pairs in length.
  • the primers may also be varied by incorporating one or more locked nucleic acids (LNA).
  • LNA are a class of nucleic acids containing altered nucleosides whose major distinguishing characteristic is the presence of a methylene bridge between the 2'-0 and 4'C atoms of the ribose ring.
  • LNA nucleosides containing the five common nucleobases that appear in DNA and RNA can base-pair with their complementary nucleosides according to Watson-Crick rules.
  • the molecular differences between normal nucleosides and LNAs give rise to differences in the stability of nucleic acid duplexes formed between LNA containing nucleic acids and non-LNA containing nucleic acids.
  • each LNA nucleotide incorporated increases the T m of a LNA/DNA nucleotide complex by 2-6°C as compared to a corresponding DNA/DNA complex.
  • the primers used in the PCR will hybridize with at least one of a nucleic acid consensus sequence for all strains of the virus (for example, CMV), a nucleic acid consensus sequence for a given strain of the virus, a sequence unique to the virus, or a sequence flanking any of the foregoing.
  • a "consensus sequence” is a sequence that is substantially conserved within a taxonomic group of viruses, such as CMV or human CMV. The sequence need not occur at the same location in the genome in each case. It is understood in the art that occasional mutations occur in consensus sequences, without negating the status of the mutant sequence as a consensus sequence.
  • silent mutation in which a nucleotide base substitution in an open reading frame does not change the amino acid specified by the corresponding mRNA codon.
  • An exemplary silent mutation is a DNA substitution from TTT to TTC. As both DNA codons encode phenylalanine, this mutation has no effect on the final protein.
  • Consensus sequences may vary at some loci by definition, in which case notations such as [CT] (either C or T), N (any base), Y (any pyrimidine), and R (any purine) are used to indicate variation.
  • CT either C or T
  • N any base
  • Y any pyrimidine
  • R any purine
  • a consensus sequence may in some cases be defined to allow a certain minimum amount of homology to a specified sequence, for example 99.9%, 99.5%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, and 70%.
  • FIG. 4 illustrates the organization of open reading frames in human CMV.
  • the CMV genome is organized as two regions of unique sequences, unique long (UL) and unique short (US), flanked by two sets of inverted repeats (TRL/IRL) and (IRS/TRS) (light shaded boxes).
  • TRL/IRL inverted repeats
  • IRS/TRS inverted repeats
  • the full genomes of at least 15 strains have been completely sequenced and are publicly available.
  • the consensus sequence can be determined by analyzing the literature of known genomic sequences of the virus or variants thereof.
  • strains for which full sequences exist include strain Towne (GenBank accession AY315197.2), strain 3157 (GenBank accession GQ221974.1), strain 3301 (GenBank accession GQ466044.1), strain AD 169 substrain varUC (GenBank accession FJ527563.1), strain AD 169 substrain varUK (GenBank accession B 000394.5), strain AF1 (GenBank accession GU179291.1), strain HAN 13 (GenBank accession GQ221973.1), strain HAN20 (GenBank accession GQ396663.1), strain HAN38 (GenBank accession GQ396662.1), strain JP (GenBank accession GQ221975.1), strain Toledo (GenBank accession GU937742.1), strain U8 (GenBank accession GU179288.1), strain Ul l (GenBank accession GU179290.1), strain VR1814 (GenBank accession GU179289.1), and strain Merlin (transgenic) (GU179001.1).
  • Sequences unique to a given virus can be identified by analyzing the literature of known genomic sequences of the virus or variants thereof. Public databases of genetic information can be queried for sequences from other sources containing regions of identity to genomic sequences of the virus. Any genomic sequences of the virus that have no identical matches can be used as unique sequences in the method.
  • Some embodiments of the unique sequence are also consensus sequences for the virus. Such sequences have the advantage of always being present in the analyte virus, but never being present in other sources of nucleic acids. This will eliminate or reduce both false negative results (when a strain of the analyte virus is present in the sample that lacks the target sequence) and false positive results (when the target sequence is present, but the analyte virus is not).
  • the target sequences for CMV include the glycoprotein B gene, the AD-1 region of glycoprotein B gene (or a nearby region), and the immediate IE-2 exon 5 gene.
  • the forward primer the major envelope glycoprotein B is AGG TCT TCA AGG AAC TCA GCA AGA (SEQ ID NO: 1)
  • the reverse primer is CGG CAA TCG GTT TGT TGT AAA (SEQ ID NO: 2).
  • forward primer for the IE-2 exon 5 gene is GAG CCC GAC TTT ACC ATC CA (SEQ ID NO: 4)
  • the reverse primer is CAG CCG GCG GTA TCG A (SEQ ID NO: 5).
  • Glycoprotein B is a highly conserved component of the complex cell-entry machinery of herpes viruses.
  • CMV the glycoprotein B gene is generally located at positions 82,066- 84,789 of the genome.
  • the general consensus sequence of the glycoprotein B gene in human CMV is shown in the attached sequence listing as SEQ ID NO: 8.
  • the antigenic determinant (AD) 1 region of the gene is generally located at positions 1-206 of SEQ ID NO: 8.
  • the target may be the glycoprotein B gene, the AD-1 region of glycoprotein B, a region nearby the AD-1 region, or a variant of either comprising conservative substitutions.
  • the target is SEQ ID NO: 8, positions 1-206 of SEQ ID NO: 8, positions 275- 346 of SEQ ID NO: 8, the glycoprotein B gene from human CMV strain Merlin (SEQ ID NO: 10), positions 1 -206 of SEQ ID NO: 10, positions 275-346 of SEQ ID NO: 10, or a variant of any of the foregoing comprising a conservative substitution.
  • the immediate early (IE) gene region of human CMV encodes the first group of proteins to be expressed after the infection of a cell. It comprises two splicing regions, IE-1 and IE-2. Exon 5 of IE-2 is highly conserved, and is generally found at positions 170,689- 174,090 in the human CMV genome.
  • the general consensus sequence of IE-2 exon 5 is provided in SEQ ID NO: 7 of the attached sequence listing.
  • the target may be IE-2 exon 5, or a variant thereof comprising conservative substitutions.
  • the target is SEQ ID NO: 7, the IE-2 exon 5 gene from human CMV strain Merlin (SEQ ID NO: 10), or a variant of any of the foregoing comprising a conservative substitution.
  • a “conservative substitution” or “conservative amino acid substitution” is a substitution that would not be expected to change the functioning of the protein product of the gene. For example, a substitution in a DNA codon that codes for the same amino acid as did the original codon (due to the degeneracy of the DNA code) is one embodiment of a conservative substitution.
  • Such a substitution may involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position.
  • any native residue in the polypeptide may also be substituted with alanine.
  • substitutions also encompass non-naturally occurring amino acid residues which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics, and other reversed or inverted forms of amino acid moieties. It will be appreciated by those of skill in the art that nucleic acid and polypeptide molecules described herein may be chemically synthesized as well as produced by recombinant means.
  • Naturally occurring residues may be divided into classes based on common side chain properties: 1) hydrophobic: norleucine, Met, Ala, Val, Leu, He; 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; 3) acidic: Asp, Glu; 4) basic: His, Lys, Arg; 5) residues that influence chain orientation: Gly, Pro; and 6) aromatic: Trp, Tyr, Phe.
  • conservative substitutions may involve the exchange of a member of one of these classes for a member from the same class.
  • Such substituted residues may be introduced into regions of the derivatives that are homologous with corresponding genes in non-human CMV orthologs, or into the non-homologous regions of the molecule.
  • hydropathic index of amino acids may be considered.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (- 0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (Kyte et al., J. Mol. Biol., 157: 105- 131, 1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within +/- 2 may be used; in an alternate embodiment, the hydropathic indices are with +/- 1 ; in yet another alternate embodiment, the hydropathic indices are within +/- 0.5.
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+- .1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (- 0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • hydrophilicity values are within +/- 2
  • the hydrophilicity values are with +/- 1
  • the hydrophilicity values are within +/- 0.5.
  • Desired amino acid substitutions can be determined by those skilled in the art at the time such substitutions are desired.
  • amino acid substitutions can be used to identify important residues of the target gene, or to increase or decrease the affinity of the target gene product with a particular binding target in order to increase or decrease the gene product's activity.
  • Exemplary amino acid substitutions are set forth in Table 1. Some embodiments of the conservative substitution will comprise any of the above exemplary substitutions. Further embodiments will comprise one or more of the preferred substitutions listed in Table 1.
  • a skilled artisan will be able to determine suitable conservative variants using well known techniques. For identifying suitable areas of the molecule that may be changed without destroying activity, one skilled in the art may target areas not believed to be important for activity. With such a comparison, one can identify residues and portions of the molecules that are conserved among similar polypeptides. One skilled in the art would also know that, even in relatively conserved regions, one may substitute chemically similar amino acids for the naturally occurring residues while retaining activity (conservative amino acid residue substitutions). Therefore, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.
  • One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of that information, one skilled in the art may predict the alignment of amino acid residues of a polypeptide with respect to its three dimensional structure. One skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test polypeptide derivatives containing a single amino acid substitution at each desired amino acid residue. The derivatives can then be screened using activity assays know to those skilled in the art and as disclosed herein. Such derivatives could be used to gather information about suitable substitution.
  • PDB protein structural data base
  • Additional methods of predicting secondary structure include “threading” (Jones, D., Curr. Opin. Struct. Biol, 7(3):377-87, 1997; Suppl et al., Structure, 4(l): 15-9, 1996), “profile analysis” (Bowie et al., Science, 253: 164-170, 1991 ; Gribskov et al, Meth. Enzym., 183: 146- 159, 1990; and Gribskov et al., Proc. Nat. Acad. Scl, 84(13): 4355.
  • More than one primer set may be utilized to increase the specificity of the method; such embodiments would be expected to produce more than one amplification product in the amplicon.
  • the second (or subsequent) forward and reverse primer may be any that are described as suitable as the first forward and reverse primers. It has been surprisingly discovered that amplifying two targets simultaneously significantly enhances the sensitivity of the method.
  • the first primer set flanks of the major envelope glycoprotein B, the AD-1 region of glycoprotein B, a region proximate to AD-1 region of glycoprotein B, or a portion thereof; and the second primer set flanks the IE-2 exon 5 gene or a portion thereof.
  • the first forward primer comprises SEQ ID NO: 1
  • the first reverse primer comprises SEQ ID NO: 2
  • the second forward primer comprises SEQ ID NO: 4
  • the second reverse primer comprises SEQ ID NO: 5.
  • the assay further comprise contacting the PCR product (amplicon) with a probe that hybridizes with at least a portion of the amplified sequence.
  • the probe comprises a nucleic acid sequence that hybridizes under highly stringent conditions with at least a portion of the amplified sequence(s) and a detectable tag. Detectable tags of various kinds are known in the art (colorimetric, fluorescent, isotopic, enzymatic, etc).
  • Some embodiments of the probe specific for the major envelope glycoprotein B comprise the nucleic acid sequence AAC CCG TCA GCC ATT CTC TCG GC (SEQ ID NO: 3).
  • Some embodiments of the probe specific for the IE-2 exon 5 gene comprise the nucleic acid sequence ACC GCA ACA AGA TT (SEQ ID NO: 6).
  • the specificity of the hybridization between the probe and the target sequence can be adjusted by increasing or decreasing the length of the probe.
  • a probe will give increased specificity and differentiation as a mismatch between the target sequence and the probe will have a significant impact on the hybridization efficiency.
  • a longer probe will provide less specificity but greater hybridization efficiency and therefore increased sensitivity.
  • the nature of the primer will influence the composition of the primer.
  • One of ordinary skill in the art would be able to alter the parameters of the probe to achieve the desired specificity and sensitivity of binding of the primer to the target sequence.
  • the probe is 10 to 50 base pairs in length.
  • the probe is 20-40 base pairs in length.
  • the probe is 14 to 23 base pairs in length.
  • the probe may also be varied by incorporating one or more locked nucleic acids (LNA).
  • LNA locked nucleic acids
  • DNA extraction generally involves lysing any cells present in the same to release chromosomal DNA, removing lipids from the sample, removing protein from the sample, and precipitating nucleic acids from solution. It has been unexpectedly discovered that the detection of viruses in saliva by the methods provided does not require the extraction of DNA from the sample prior to amplification. This has the advantage of not requiring the time-consuming and laborious steps of DNA extraction.
  • Kits for use with the assays comprising a polymerase, a forward primer, and a reverse primer.
  • Embodiments of the kit may further comprise a probe.
  • Further embodiments comprise at least one of a reagent for visualizing the probe (such as a substrate of horseradish peroxidase, or a radio-scintillation cocktail), or a source of illumination (such as an ultraviolet lamp) for visualizing the probe.
  • the primers and probe may be any that are disclosed as suitable for the method.
  • the polymerase may be any DNA polymerase, but it is preferably a DNA polymerase that is active at thermophilic temperatures to facilitate the rapid denaturation and hybridization of nucleic acids that occurs during PCR.
  • thermophilic polymerases are well known in the art, and examples include DNA polymerase from the bacterium Thermus aquaticus.
  • the kit may be for the detection of a virus in a saliva sample; in an exemplary embodiment the kit is for the detection of human CMV in a saliva sample. In further embodiments, the kit is for the diagnosis of CMV infection.
  • the kit is for the detection of a virus in saliva by real-time PCR, and includes no reagents for DNA extraction.
  • the kit comprises a first forward primer, a first reverse primer, a second forward primer, and a second reverse primer.
  • Some embodiments of the probe are specific to at least one of the major envelope glycoprotein B gene and the immediate early region 2 exon 5.
  • Specific embodiments of the probe comprise at least one of SEQ ID NO: 3 and SEQ ID NO: 6.
  • the primers hybridize under highly stringent conditions to sequences flanking the major envelope glycoprotein B gene, the AD-1 region, a region proximate to the AD-1 region, or a portion thereof.
  • the forward primer comprises SEQ ID NO: 1 and the reverse primer comprises SEQ ID NO: 2.
  • the probe comprise SEQ ID NO: 3.
  • the primers hybridize under highly stringent conditions to sequences flanking IE2 exon 5 or a portion thereof.
  • the forward primer comprises SEQ ID NO: 4 and the reverse primer comprises SEQ ID NO: 5.
  • the kit of claim 40 further comprises a probe comprising SEQ ID NO: 6.
  • kits comprises a first set of primers that hybridize under highly stringent conditions to the major envelope glycoprotein B gene or a portion thereof; a second set of primers that hybridize under highly stringent conditions to sequences flanking the IE2 exon 5 or a portion thereof; a polymerase; and lacks reagents for DNA extraction from the sample.
  • Saliva specimens were collected by swabbing the inside of the baby's mouth using a sterile polyester fiber-tipped applicator (PurFybr Inc., Munster, IN) and transported to the Central Laboratory at the University of Alabama at Birmingham within one week of collection. 14 ' 19 During Phase I, saliva swabs were placed in TM and tested by rapid culture and liquid saliva PCR. During Phase II, an additional saliva swab collected at the same time was allowed to air dry, placed in a sterile tube without TM ("dried saliva swab”), and maintained and transported at ambient temperature to the Central Laboratory on a weekly basis.
  • TM sterile polyester fiber-tipped applicator
  • saliva specimens from some of the enrolled infants were tested using all three methods (rapid culture, liquid saliva PCR, and dried saliva PCR). The screening saliva specimens collected during Phase II were tested by rapid culture and dried saliva PCR only.
  • Specimen Processing Liquid saliva specimens were processed as described. 14 ' 19 Dried saliva specimens were processed by adding 300 ⁇ of PCR- grade water to the tubes containing saliva swabs, vortexing, and incubating for 20 minutes at room temperature. Five iL of the TM or the eluate containing saliva was used without a DNA extraction step for real-time PCR.
  • Detection of CMV in Saliva Specimens Using the Rapid Culture Assay A rapid culture assay for the detection of early antigen fluorescent foci using a monoclonal antibody against the major immediate early antigen of CMV was used to detect CMV in saliva specimens. 14 ' 18 ' 19 Laboratory personnel performing the rapid culture were blinded to the results of PCR and vice versa.
  • Real-Time PCR of Saliva Specimens A real-time PCR protocol using the ABI 7500 Real Time PCR System (Applied Biosystems Inc., Foster City, CA) and utilizing Absolute Low ROX QPCR mix (ABGene USA, Rockford, IL) was performed to detect CMV DNA in saliva samples.
  • the real-time PCR protocol was described in a recent report 14 , which is incorporated by reference herein to teach this method.
  • lyophilized Primers 80,000 pmol (ABI, Cat #4304971), first probe (described below) 50,000 pmol (ABI, Cat# 4316032), second probe (described below) 50,000 pmol (ABI, Cat # 450003), Absolute Low ROX QPCR mix (ABgene, Cat #AB1318/B), molecular grade PCR water, 5-Prime (Fisher, Cat #955155017), optical PCR plates (ABgene, Cat #AB-1100/150), adhesive film for QPCR (ABgene, Cat #AB-1170), QPCR DNA standards at 10, 100, 1000, 10,000, and 100,000 copies/5 iL with target regions for both primer/probe sets.
  • the final concentrations of primers and probe to be used in the PCR reaction were 900 nM and 200 nM, respectively. All samples were run in duplicate. Each panel contained a no-template control (NTC). Standard curves were derived from serial 10- fold dilutions of a positive control template (Calibrated Plasmid DNA). The positive control template was derived from cloning PCR target sequence into TOPO TA pCR2.1 (Invitrogen, CA) plasmid.
  • the cycling parameters were as follows: Taq polymerase activation at 95° C for 15 minutes, denaturation at 95° C for 15 seconds, annealing/extension at 60° C for 1 minute, all performed for 40 cycles; measurement of fluorescence was performed at the end of each annealing/extension cycle.
  • Two CMV primer sets from highly conserved target regions one targeting the major envelope glycoprotein B (forward primer AGG TCT TCA AGG AAC TCA GCA AGA, reverse primer CGG CAA TCG GTT TGT TGT AAA and probe 6FAM - AAC CCG TCA GCC ATT CTC TCG GC - TAMRA) and a second primer set from the IE-2 exon 5 gene (forward primer GAG CCC GAC TTT ACC ATC CA, reverse primer CAG CCG GCG GTA TCG A and probe VIC - ACC GCA ACA AGA TT - MGBNFQ) were included in the PCR reaction.
  • the reaction mixture contained primers at a concentration of 900 nM and the probe at 250 nM concentration.
  • Each 25 iL reaction mixture contained 20 iL of master mix and 5xL of test sample. Reactions were performed in duplicate and each plate contained plasmid standards incorporating both target sequences in 10-fold dilutions ranging between 100,000 and 10 genomic equivalents (ge) per reaction to generate standard curves. A sample was considered positive if > ge per reaction on the final PCR run.
  • Positive LR was sensitivity/(l -specificity) and the negative LR was (l-sensitivity)/specificity and confidence intervals were determined using the method described by Simel and colleagues. All statistical analyses were performed using SAS software version 9.2 (SAS Institute, Inc., Cary, NC).
  • Study Population and Specimens During the study period, 34,989 infants were enrolled. The mean (SD) age at collection of saliva specimens for rapid culture and PCR was 1.0 (1.2) day. Characteristics of the study population are shown in Table 2. Infants were screened predominantly (98.0%) from the well baby nurseries. The median age for collection of follow-up samples was 3.6 weeks with an interquartile range of 2.6 to 6.6 weeks. Overall, 177 newborns (0.51 %; 95% CI, 0.43-0.59%) tested positive for CMV on screening by rapid culture and/or PCR of saliva. No study-related adverse events were observed.
  • the positive LR for the liquid saliva PCR assay was 2197 (95% CI, 1099-4393) and the negative LR was 0 (95% CI, 0-0.1).
  • 79 (85%) infants were enrolled in follow-up.
  • 72 infants who were positive by both rapid culture and PCR and enrolled in follow-up only one infant was negative on retesting.
  • eight PCR-only positive infants seven were enrolled in follow-up; of those, six were negative by rapid culture and PCR of both saliva and urine specimens.
  • the sensitivity and the specificity of the dried saliva PCR assay were 97.4% (74/76, 95% CI, 90.8-99.7%) and 99.9% (17245/17253, 95% CI, 99.9- 100%), respectively.
  • the positive and negative predictive values for the dried saliva PCR were 90.2% (74/82, 95% CI, 81.7-95.7%) and 99.9% (17243/17245, 95% CI, 99.9-100%), respectively.
  • the positive LR for the dried saliva PCR assay was 2100 (95% CI, 1049-4202) and the negative LR was 0.03 (95% CI, 0.01 -0.10) (Table 3).
  • the dried saliva PCR assay failed to detect two CMV-infected newborns leading to slightly lower sensitivity (97.4%, 95% CI, 90.8%-99.7%) than the liquid saliva PCR.
  • the simplified specimen collection, storage, and transport procedures combined with high sensitivity of dried saliva PCR confirm this method as a reasonable newborn CMV screening approach.
  • the need for collection of an additional specimen adds to the complexity of the existing newborn screening programs, the saliva PCR assays reported in this study have several advantages for newborn CMV screening. These include: (1) reasonable sensitivity and specificity, (2) non-invasive specimen collection, (3) elimination of the DNA extraction step, which simplifies the laboratory procedures providing significant cost savings, and (4) dried saliva specimens can be stored and transported at room
  • Yamamoto AY Mussi-Pinhata MM, Marin LJ, Brito RM, Oliviera PF, Coelho TB. Is saliva as reliable as urine for detection of cytomegalovirus DNA for neonatal screening of congenital CMV infection? J Clin Virol 2006;36:228-30. 13. Yamamoto AY, Mussi-Pinhata MM, Pinto PCG, Figueriedo LTM, Jorge SM.
  • Boppana SB, Ross SA, Novak Z, et al. Dried blood spot real-time polymerase chain reaction assays to screen newborns for congenital cytomegalovirus infection. JAMA 2010;303:1375-82.
  • CMV cytomegalovirus
  • Table 3 Utility of real-time PCR assays of liquid and dried saliva specimens in identifying infants with congenital CMV infection.
  • PCR polymerase chain reaction
  • CMV cytomegalovirus
  • PCR polymerase chain reaction
  • CMV cytomegalovirus

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Abstract

L'invention se rapporte aux infections congénitales à cytomégalovirus (CMV) qui sont une cause de mortalité importante chez les jeunes enfants et les adultes immunodéprimés. La plupart des jeunes enfants infectés par le CMV ne sont pas rapidement identifiés en raison de l'absence de symptômes. On a découvert qu'une analyse basée sur l'amplification en chaîne par polymérase (PCR) destinée à tester des échantillons de salive pour y détecter le CMV était rapide, précise et peu coûteuse. Certaines versions de l'analyse utilisent des amorces spécifiques aux séquences flanquant la glycoprotéine B principale de l'enveloppe hautement conservée ou les gènes à exon 5 de région 2 précoces immédiats, hautement conservés. L'analyse supplante la technique standard de culture rapide en termes de précision, de rapidité et d'économie. Lorsque l'analyse est effectuée sur de la salive séchée, sa précision n'est pas fortement dégradée et elle est étonnamment bien plus sensible qu'une analyse par PCR avec du sang séché. Cette analyse permettra un plus large éventail de tests pour détecter et traiter les infections congénitales à CMV, ce qui évitera potentiellement de nombreux cas de maladies associées.
PCT/US2011/034809 2010-04-30 2011-05-02 Analyse par amplification en chaîne par polymérase d'un échantillon de salive pour détecter une infection à cytomégalovirus WO2011137445A2 (fr)

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FIDOUH-HOUHOU, N. ET AL.: 'Salivary cytomegalovirus (CMV) shedding, glycoprotein B genotype distribution, and CMV disease in human immunodeficiency virus-seropositive patients.' CLINICAL INFECTIOUS DISEASES. vol. 33, no. 8, 15 October 2001, pages 1406 - 1411 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9206468B2 (en) 2010-12-17 2015-12-08 Wisconsin Alumni Research Foundation One-step method of elution of DNA from blood samples
WO2013090040A1 (fr) * 2011-12-16 2013-06-20 Wisconsin Alumni Research Foundation Détection de l'adn du cytomégalovirus au moyen d'amplification à partir d'échantillons de sang
CN105331698A (zh) * 2015-11-05 2016-02-17 瀚吉康生物科技（北京）有限公司 多重分型检测人巨细胞病毒(hcmv)的试剂盒及其应用

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